Simvastatin Improves Microcirculatory Function in Nonalcoholic Fatty Liver Disease and Downregulates Oxidative and ALE-RAGE Stress

Increased reactive oxidative stress, lipid peroxidation, inflammation, and fibrosis, which contribute to tissue damage and development and progression of nonalcoholic liver disease (NAFLD), play important roles in microcirculatory disorders. We investigated the effect of the modulatory properties of simvastatin (SV) on the liver and adipose tissue microcirculation as well as metabolic and oxidative stress parameters, including the advanced lipoxidation end product–receptors of advanced glycation end products (ALE-RAGE) pathway. SV was administered to an NAFLD model constructed using a high-fat–high-carbohydrate diet (HFHC). HFHC caused metabolic changes indicative of nonalcoholic steatohepatitis; treatment with SV protected the mice from developing NAFLD. SV prevented microcirculatory dysfunction in HFHC-fed mice, as evidenced by decreased leukocyte recruitment to hepatic and fat microcirculation, decreased hepatic stellate cell activation, and improved hepatic capillary network architecture and density. SV restored basal microvascular blood flow in the liver and adipose tissue and restored the endothelium-dependent vasodilatory response of adipose tissue to acetylcholine. SV treatment restored antioxidant enzyme activity and decreased lipid peroxidation, ALE-RAGE pathway activation, steatosis, fibrosis, and inflammatory parameters. Thus, SV may improve microcirculatory function in NAFLD by downregulating oxidative and ALE-RAGE stress and improving steatosis, fibrosis, and inflammatory parameters.


Biochemical Parameters
Commercial kits were used to measure liver and serum cholesterol, triglycerides, HDL and LDL, as well as the enzymatic activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (Bioclin System II, Belo Horizonte, MG, Brazil). Fasting blood glucose levels were measured using an automated glucose monitor (One Touch Ultra2, Johnson & Johnson Medical S.A., Buenos Aires, Argentina).

Oral Glucose Tolerance Test (OGTT)
An oral glucose tolerance test (OGTT was performed on mice fasted for 6 h. Blood glucose levels were measured at 0, 15, 60, and 120 min following oral glucose overload (2 g/kg), and areas under the glucose curves (AUCs) were determined. Blood glucose levels were measured using an automated glucose monitor (One Touch Ultra2, Johnson & Johnson Medical S.A., Buenos Aires, Argentina).

Measurement of Hepatic Nitrite (NO 2-) Concentration
NO production in the liver tissue was quantified by measuring the released nitric oxide (NO) metabolite, nitrite, using Griess reagent (modified, Sigma G4410, St. Louis, MO, USA) according to the manufacturer's instructions. Nitrite levels were determined using a standard curve prepared against a standard solution of NaNO 2 [35].

Histopathology
Fragments of the left lateral lobe of the liver were fixed in Millonig's phosphatebuffered formalin solution (pH 7.2) and embedded in paraffin for light microscopic analysis (Nikon, model 80i and DSRi1 digital camera, Nikon Instruments, Inc., Melville, NY, USA). Histological features of hematoxylin and eosin (H&E)-stained sections were classified into the following categories: Steatosis, inflammation, and hepatocellular damage. Each sample was analyzed using the stereological method of grid-point counting which measures the fatty content of the liver (STEPanizer stereology version 1, Bern, Switzerland) [36,37]. An NAFLD activity score (NAS) was used to classify the stage of liver disease, providing an overall diagnosis for each case as NASH (score ≥ 5), borderline (score 3-4) or not NASH (score 0-2) [38]. Masson's trichrome stain was used to detect and evaluate fibrosis by collagen quantification using ImageJ (ImageJ version 1.53e, Madison, WI, USA) [39]. For each parameter, five histological sections from six mice per group were analyzed.

Immunohistochemistry
Immunohistochemical analysis of hepatic alpha smooth muscle actin (α-SMA) was performed to determine HSC activation. Briefly, following deparaffinization, sections were treated with 3% hydrogen peroxide (H 2 O 2 ) and blocked using non-fat dry milk and bovine serum albumin (BSA). Sections were rinsed in PBS and incubated overnight in a humidified chamber at 4 • C with anti-α-SMA monoclonal primary mouse antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a dilution of 1:500. The slides were then washed in PBS and treated with a biotinylated secondary antibody, followed by incubation with streptavidin peroxidase, both for 30 min at room temperature. Diaminobenzidine (DAB) was used as the chromogen. Slides were counterstained with Mayer's hematoxylin.

RT-PCR
Total RNA was extracted from the middle lobe of the liver using the RNeasy Mini Kit (Qiagen, Düsseldorf, Germany). Next, cDNA synthesis was performed using 1 µg RNA in a final volume of 20 µL via a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Beverly, MA, USA). The primers used for product amplification were of liver-type fatty acid-binding protein (L-FABP), forward 5 -CCATGACTGGGGAAAAAGTC-3 and reverse 5 -GCCTTTGAAAGTTGTCACCAT-3 ; collagen type I alpha 1 (COL1A1), forward 5 -CTCCTGGCAAGAATGGAGAT-3 and reverse 5 -AATCCACGAGCACCCTGA-3 ; and β-actin forward 5 -AGATTACTGCTCTGGCTCCTAGC-3 and reverse 5 -ACTCATCGTACT CCTGCTTGCT-3 . RT-PCR was performed using a Power SYBR Green PCR Master Mix (Applied Biosystems, Beverly, MA, USA) according to the manufacturer's instructions and run on a 7500 Fast platform (Applied Biosystems, Beverly, MA, USA). The relative expression of the genes of interest was calculated using the ∆∆Ct method and normalized to β-actin expression [17].

Antioxidant Enzyme Activity
Superoxide dismutase (SOD) activity was measured indirectly via the SOD inhibition index using a spectrophotometer at a wavelength of 530 nm [41]. In order to evaluate catalase (CAT) activity, the consumption of H 2

Intravital Microscopy
For intravital microscopy, mice fasted overnight were anesthetized with ketamine/xylazine (100 mg/kg and 10 mg/kg, respectively, intraperitoneally). After laparotomy, the left liver lobe and epididymal fat were externalized for assessment of microcirculation using intravital microscopy (Olympus BX150WI; Center Valley, PA, USA), as described previously [17]. After intravenously administering 0.3 mg/kg rhodamine 6G (Sigma Chemical Co., St. Louis, MO, USA), leukocyte-endothelial interaction was assessed by counting the number of labeled leukocytes rolling or adhering to hepatic or adipose tissue microcirculation. In the liver, the number of vitamin A-positive hepatic stellate cells (HSCs) was determined as the number of fluorescent cells derived from vitamin A autofluorescence. After intravenously administering 0.05 mL of 2% fluorescein isothiocyanate (FITC)-labeled dextran (molecular weight 150,000; Sigma Chemical Co., St. Louis, MO, USA), hepatic microcirculation images were acquired (Prime Intervision, Doral, FL, USA). Sinusoidal density analysis was performed using ImageJ software (ImageJ 1.47 v; Wayne Rasband, National Institute of Health, Bethesda, MD, USA) to determine the mean value of functional capillary density.

Quantification of Advanced Lipoxidation End Products (ALEs)
Concentrations of fluorescent ALEs in liver tissue were determined as previously described [17]. Briefly, on a SpectraMax M5 ELISA Microplate Reader (Molecular Devices, San Jose, CA, USA), ALEs were measured at a protein concentration of 1 kg/m 3 at an emission wavelength of 440 nm and an excitation wavelength of 370 nm. A native BSA preparation (1 kg/m 3 , 0.1 mol/m 3 NaOH) served as reference, and its fluorescence intensity was determined as the unit of fluorescence. The fluorescence values of the samples were expressed in arbitrary units (AUs) [17,43].

Western Blot
Western blotting was used to examine the protein expression of inducible nitric oxide synthase (iNOS) and the receptor for AGE (RAGE). All primary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, Santa Cruz, CA, USA) and used at a dilution of 1:500. Liver tissue was homogenized in lysis buffer (20 mmol/m 3 Trizma/137 mmol/m 3 NaCl/10% glycerol/1% Nonidet p-40/2 mmol/m 3 EDTA) and centrifuged for 45 min at 14,000× g at 4 • C. Protein content was determined via the bicinchoninic acid method using BSA (Sigma-Aldrich, St. Louis, MO, USA) as a standard (BCA Protein Assay Kit, Thermo Scientific, Waltham, MA, USA). Next, 30 µg of protein per lane was separated on a 12% sodium dodecyl sulfate gel and transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad Laboratories, Munich, Germany). After blocking with 5% BSA, the membranes were incubated overnight at 4 • C with primary antibodies. A secondary RDye 680RD donkey-anti-goat antibody (1:15,000; LI-COR Biosciences, Cambridge, UK) for iNOS and a secondary RDye 680RD donkey anti-goat antibody (1:15,000; LI-COR Biosciences, Cambridge, UK) for RAGE were used for detection. The bound complex was detected using the Odyssey Infrared Imaging System (Li-Cor; Lincoln, NE, USA). Images were analyzed using Image Studio Lite software version 4.0.21 (Li-Cor, Lincoln, NE, USA) to obtain integrated intensities and normalized with respect to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) signals (mouse anti-GAPDH monoclonal antibody, 1:30,000; Fitzgerald Industries International, Acton, MA, USA), followed by a secondary IRDye 800CW goat anti-mouse antibody (1:20,000; LI-COR Biosciences).

Statistical Analysis
Results are expressed as the mean ± SEM of each group. Normality was assessed using the Shapiro-Wilk test, and comparisons between groups were made using two-way analysis of variance (ANOVA) followed by Tukey's post hoc test. The relationship between ALEs and metabolic and microcirculatory parameters was examined using Pearson's correlation coefficient and linear regression. Data were analyzed using GraphPad Prism 8.0.1 (GraphPad Software Inc., La Jolla, CA, USA) and R software (version 3.4.2; R Core Team, R Foundation for Statistical Computing, Vienna, Austria). Statistical significance was set at p < 0.05.

SV Protects against NAFLD-Induced Metabolic Changes
To explore the effects of SV on fatty liver disease, mice were fed either control or HFHC purified diets and subgroups of control and HFHC-fed mice were treated with SV ( Figure 1A). All parameters were similar in the groups of mice fed with normal chow. Both HFHC-fed and HFHC+SV mice gained more weight than the controls fed normal chow (p < 0.001); ( Figure 1B). HFHC-fed as well as HFHC+SV mice displayed impaired glucose tolerance (p < 0.05) compared to the controls ( Figure 1C). HFHC-fed and HFHC+SV mice showed significantly higher fat depots compared to those of the controls ( Figure 1D). All groups had similar liver weights and liver/body weight ratios ( Figure 1E). Furthermore, HFHC-fed mice showed significantly increased hepatic cholesterol and triglycerides, serum cholesterol (total and LDL), and triglycerides, as well as ALT and AST enzyme activity and a reduction in HDL cholesterol, compared to those of control mice ( Figure 1F). Histologically, steatosis ( Figure 2A,B), hepatocyte ballooning (Figure 2A), and fibrosis ( Figure 2E,F) were observed in the livers of HFHC-fed mice, while no inflammatory cell infiltration was observed ( Figure 2A). Accordingly, steatosis, hepatocyte ballooning, and fibrosis were significantly higher in HFHC-fed vs. control and HFHC+SV mice livers, contributing to significantly higher NAFLD activity scores in HFHC-fed mice livers (all p < 0.001; Figure 2C). HFHC-fed mice showed increased mRNA transcripts of L-FABP in the liver ( Figure 2D). 2A), and fibrosis ( Figure 2E,F) were observed in the livers of HFHC-fed mice, while no inflammatory cell infiltration was observed ( Figure 2A). Accordingly, steatosis, hepatocyte ballooning, and fibrosis were significantly higher in HFHC-fed vs. control and HFHC+SV mice livers, contributing to significantly higher NAFLD activity scores in HFHC-fed mice livers (all p < 0.001; Figure 2C). HFHC-fed mice showed increased mRNA transcripts of L-FABP in the liver ( Figure 2D).

SV Prevents Microcirculatory Dysfunction in HFHC-Fed Mice
The effects of SV on microcirculatory parameters were assessed using intravital microscopy and LSCI flowmetry. HFHC-fed mice displayed significantly higher numbers of rolling and adherent leukocytes in hepatic and adipose microcirculation than the controls fed with normal chow, but this was not observed in HFHC+SV-fed mice ( Figure 3A,B). HSC activation in HFHC-fed mice was increased compared to that of control mice fed with normal chow (both p < 0.001), as evidenced by increased α-SMA protein expression in the liver tissue ( Figure 3C,D) and decreased vitamin A-positive cell numbers (p < 0.001) ( Figure 3E,F); however, such an increase was not observed in HFHC+SV mice. Accordingly, HFHC-fed mice showed increased mRNA transcripts of collagen 1α compared to control and HFHC-treated mice, as indicated by qPCR analysis ( Figure 2G). Furthermore, the hepatic capillary network architecture as well as its vascular density was altered in HFHC-fed mice, as indicated by a 30% reduction in sinusoid density and distorted sinusoidal network, compared to those of control and HFHC+SV mice ( Figure 3G,H).

SV Prevents Microcirculatory Dysfunction in HFHC-Fed Mice
The effects of SV on microcirculatory parameters were assessed using intravital microscopy and LSCI flowmetry. HFHC-fed mice displayed significantly higher numbers of rolling and adherent leukocytes in hepatic and adipose microcirculation than the controls fed with normal chow, but this was not observed in HFHC+SV-fed mice ( Figure 3A,B). HSC activation in HFHC-fed mice was increased compared to that of control mice fed with normal chow (both p < 0.001), as evidenced by increased α-SMA protein expression in the liver tissue ( Figure 3C,D) and decreased vitamin A-positive cell numbers (p < 0.001) ( Figure 3E,F); however, such an increase was not observed in HFHC+SV mice. Accordingly, HFHC-fed mice showed increased mRNA transcripts of collagen 1α compared to control and HFHC-treated mice, as indicated by qPCR analysis ( Figure 2G). Furthermore, the hepatic capillary network architecture as well as its vascular density was altered in HFHC-fed mice, as indicated by a 30% reduction in sinusoid density and distorted sinusoidal network, compared to those of control and HFHC+SV mice ( Figure 3G,H).
Basal hepatic and adipose tissue microvascular blood flow was significantly decreased in HFHC-fed mice compared with that of the control or HFHC+SV groups ( Figure 4A,B). Response of the adipose tissue endothelium-dependent vasodilator to Ach in HFHC mice was impaired in a dose-dependent manner, compared to that in normal chow-fed or HFHCfed mice ( Figure 4C). Basal hepatic and adipose tissue microvascular blood flow was significantly decreased in HFHC-fed mice compared with that of the control or HFHC+SV groups ( Figure  4A,B). Response of the adipose tissue endothelium-dependent vasodilator to Ach in HFHC mice was impaired in a dose-dependent manner, compared to that in normal chow-fed or HFHC-fed mice ( Figure 4C).

SV Protects against NAFLD-Induced Oxidative Damage and Inflammation
Examination of hepatic and adipose tissue oxidative stress via TBARs revealed that lipid peroxidation in HFHC-fed mice was significantly increased compared to that in the control or the HFHC+SV group, (Figure 5A,D). The activity of the antioxidant catalase (CAT) was significantly decreased (Figure 5B), while that of superoxide dismutase (SOD) was increased ( Figure 5C) in the liver of HFHC-fed mice, compared to that of the control or HFHC-treated group. The adipose tissue of HFHC mice showed increased CAT activity ( Figure 5E) and decreased SOD activity ( Figure 5F) compared to that of control mice fed with normal chow and HFHC+SV. ALE deposition in the liver tissues of HFHC-fed mice was increased, but such an increase was not observed in normal chow-fed mice or HFHC+SV mice ( Figure 5G). Similarly, RAGE protein expression was significantly increased in HFHCfed mice compared to that in the control and HFHC+SV groups ( Figure 5H). Furthermore, increases in hepatic nitric oxide bioavailability ( Figure 5I) and iNOS protein expression ( Figure 5J) were observed in mice fed with HFHC diet compared to those fed with normal chow or HFHC+SV mice.
Hepatic ALE deposition correlated with RAGE and microcirculatory parameters. A strong positive correlation was observed between hepatic ALE levels and hepatic RAGE protein expression levels and hepatic rolling of leukocytes. The analysis also demonstrated a moderate correlation with hepatic adhesion of leukocytes ( Figure 5K). Pearson's correlation coefficient showed a strong negative correlation between hepatic ALE deposition and

SV Protects against NAFLD-Induced Oxidative Damage and Inflammation
Examination of hepatic and adipose tissue oxidative stress via TBARs revealed that lipid peroxidation in HFHC-fed mice was significantly increased compared to that in the control or the HFHC+SV group, (Figure 5A,D). The activity of the antioxidant catalase (CAT) was significantly decreased ( Figure 5B), while that of superoxide dismutase (SOD) was increased ( Figure 5C) in the liver of HFHC-fed mice, compared to that of the control or HFHC-treated group. The adipose tissue of HFHC mice showed increased CAT activity ( Figure 5E) and decreased SOD activity ( Figure 5F) compared to that of control mice fed with normal chow and HFHC+SV. ALE deposition in the liver tissues of HFHC-fed mice was increased, but such an increase was not observed in normal chow-fed mice or HFHC+SV mice ( Figure 5G). Similarly, RAGE protein expression was significantly increased in HFHC-fed mice compared to that in the control and HFHC+SV groups ( Figure  5H). Furthermore, increases in hepatic nitric oxide bioavailability ( Figure 5I) and iNOS protein expression ( Figure 5J) were observed in mice fed with HFHC diet compared to those fed with normal chow or HFHC+SV mice.

Discussion
While the protective effects exerted by SV against NAFLD are documented [23,31,44], information regarding the mechanisms underlying such protection is scant. In this study, we demonstrate, for the first time, that SV protects against high-fat-high-carbohydrate diet-induced hepatic and adipose tissue microcirculatory dysfunction. Furthermore, we show that amelioration of microcirculatory disturbances was associated with decreased lipid peroxidation and ALE-RAGE axis activation, suggesting that SV contributes to microcirculatory recovery by modulating oxidative and ALE-RAGE stress.
As previously evidenced during pre-clinical and clinical studies, SV improves serum and hepatic lipid profiles and liver transaminases, thus impairing steatosis and NAFLD progression [45][46][47][48][49][50][51]. Management of dyslipidemia, liver function and steatosis is considered crucial for attenuating the progression of NAFLD [52]. Furthermore, SV decreased L-FABP expression. L-FABP mRNA is highly expressed in HFHC-fed mice, and such increased expression is associated with steatosis, as L-FABP plays a role in lipid and fatty acid metabolism [53]. L-FABP knockout protects Western diet-fed mice from developing hepatic steatosis and fibrogenesis [54][55][56]. Previously, Landrier et al. showed that SV treatment induced a dose-dependent increase in L-FABP mRNA levels in isolated rat hepatocytes and in vivo [57]. Regarding their in vitro results, the complex intracellular cross-talk and lipid signalling that occurs in vivo between hepatocytes and other cells such as HSCs should not be neglected, underscoring the importance of in vivo models for understanding complex systems [56]. Regarding the in vivo results, Landrier and colleagues used only three rats/group and a statin dose of 100 mg/kg for five consecutive days, which is a very high and unusual dose and a very short treatment duration. In addition, the differences in the protocol between Landrier and colleagues and ours make comparative analysis difficult. In another study, Song et al. showed that there was no significant upregulation of lipid transporter genes such as CD36 and FABPs in hepatocytes treated with lovastatin or SV [58]. Therefore, the effects of statins on genes involved in lipid metabolism are still controversial and require further studies.
A major objective of this study was to investigate the pathophysiological processes that occur in the microcirculation of mice with NAFLD, as well as the potential of simvastatin to modulate these processes. The unique anatomical relationship between the liver and adipose tissue, which are connected by the portal circulation, indicates that studying the hepatic and visceral adipose tissue microcirculation is clinically relevant. Thus, we utilized in vivo imaging techniques to analyze the microcirculation of epididymal fat deposits, as being representative of visceral adipose tissue (VAT), as well as hepatic microcirculation. Consistent with previous studies, we showed that the liver of mice with NASH undergo (a) a substantial decrease in capillary density, (b) decreased basal blood flow in the hepatic microcirculation, (c) a pronounced increase in HSC activation, and (d) increased leukocyte recruitment in the sinusoidal and post-sinusoidal venules [17,20,59]. Furthermore, SV rescued liver microcirculation alterations induced by NAFLD. Decreased lipid accumulation and steatosis induced by SV may reduce the narrowing of sinusoids in the hepatocytes of NAFLD mice and thus promote blood flow recovery in microcirculation [17,18,60]. Moreover, as activated HSCs play a pivotal role in collagen deposition as well as in vascular tone regulation [61,62], the protective effect exerted by SV against HSC activation may significantly impact fatty liver progression [45]. Our analysis showed decreased fibrosis and positive α-SMA staining, in addition to a reduction in collagen I gene expression in the livers of HFHC-fed mice treated with SV. Substantiating this, Gracia-Sancho et al. demonstrated that SV maintains HSCs in a quiescent state in a cirrhotic liver and thereby significantly reduces liver fibrosis [63]. Bravo and coworkers demonstrated in sorted cells from rats with NASH that the vasoprotective effect of statins was associated with regression of HSC activation and restoration of LSEC differentiation [64]. In addition, accumulations of circulating leukocytes, that are mechanically trapped in or adhered to the vasculature, may further exacerbate sinusoid compression [20]. Thus, SV may protect the microcirculation by decreasing leukocyte recruitment [65]. SV improved vascular disturbances and ameliorated portal hypertension in carbon tetrachloride-induced cirrhotic rats, by increasing nitric oxide bioavailability and improving liver sinusoidal endothelial function [66]. In cirrhotic livers, SV enhanced KLF2-dependent vasoprotective mechanisms, thereby preventing liver damage, inflammation and oxidative stress and improving endothelial dysfunction [67]. This was also observed in explanted rat livers when SV was added to the storage solution [68]. The observed reversal of functional capillary density in the hepatic microcirculation by SV may be a consequence of the decrease in (i) steatosis, (ii) fibrosis, (iii) HSC activation and COL1A1 expression and (iv) leukocyte recruitment, which in turn reversed tissue hypoxia and recovered microvascular blood flow.
Our analysis of VAT microcirculation revealed that mice fed with an HFHC diet exhibited endothelial dysfunction, as evidenced by an impaired vasodilatation response to Ach. Similarly, mice with NAFLD showed reduced blood perfusion and increased leukocyte recruitment in the VAT microvasculature. While studies have demonstrated leukocyte infiltration of adipose tissue in obese mice and humans [69,70], to our knowledge, ours is the first study to evaluate VAT vascular beds in NAFLD. As body weight increases, adipose tissue expands via hypertrophy or hyperplasia (increased adipocyte size or increased adipocyte number, respectively). However, hypertrophy is not accompanied by an increase in capillary number, and may result in adipose tissue hypoxia, tissue remodeling, leukocyte recruitment, and tissue inflammation [71][72][73][74]. Crosstalk between adipose tissue and the liver may further exacerbate the pathophysiological processes associated with NAFLD [75]. Adipose tissue hypertrophy and hyperplasia increase the release of non-esterified free fatty acids (FFAs) and proinflammatory cytokines into the circulation, resulting in ectopic lipid deposition, low-grade chronic inflammation, and insulin resistance [76]. Concomitantly, adipose tissue-derived factors may be released into the circulation and target the liver, thereby aggravating liver injury [77,78] and subsequently impairing microcirculation function. Notably, microcirculation dysfunction in the VAT was fully recovered by SV.
In order to elucidate the mechanisms by which SV protects against hepatic and VAT microcirculatory dysfunction, we analyzed alterations in the balance between pro-oxidant and antioxidant factors in these tissues due to lipid peroxidation, antioxidant enzyme activities, and the ALE-RAGE axis. We further observed that an increase in hepatic and VAT MDA levels, which caused an increase in lipid oxidation in NAFLD-induced mice, acted as a key insult contributor to the progression of NAFLD. Concomitantly, the first line of the antioxidant defense mechanism, SOD and CAT, which protects the organism from ROS-mediated oxidative damage, were altered in the liver and VAT of mice with NAFLD, as was previously reported [79][80][81]. Increased antioxidant enzyme activity may indicate an adaptive response that counteracts an increase in lipid peroxidation and ROS in NAFLD because increased oxidative stress in the tissues causes cell damage, induces cytokine production, and activates inflammatory cells, which further stimulates ROS formation [82,83]. As NAFLD progresses, antioxidant reserves may be depleted, resulting in a lower antioxidant capacity. Previous studies have demonstrated that increased oxidative stress pathway activation is associated with reduced microvascular blood flow [17,42,84,85]. Notably, we showed that SV restored the oxidative balance in mice fed with an HFHC diet. Statins reduce the production of superoxide anions via NADPH oxidase [86] by directly affecting this enzyme complex [87] which leads to the inhibition of Rac1 prenylation [88]. Inhibition of protein prenylation by SV results in the modulation of inflammatory and oxidative responses [89]. In addition, SV activates antioxidant enzymes via Nrf2, a key regulator of the antioxidant defense machinery [44,90].
We found that SV therapy reversed a significant increase in ALE deposition and RAGE protein expression in hepatic tissues. Lipid peroxidation, induced by oxidative stress, causes an increase in the endogenous production of reactive carbonyl species (RCS) and their derivatives, such as MDA, giving rise to ALEs such as MDA-Lys, HNE-Lys, FDP-Lys, carboxymethyl-lysine (CML), and S-carboxymethyl-cysteine, among others [91]. ALEs regulate a multitude of cell signaling pathways and play a role in a wide range of molecular and biological processes, ranging from protein, DNA, and phospholipid damage to signaling pathway activation and/or modification in response to oxidative stress stimuli [92,93]. ALEs exert their pathogenic effects by binding to receptors of advanced glycation end products (AGEs), termed RAGEs, which increases inflammatory, oxidative, and fibrotic responses [94]. Endothelial cells, vascular smooth muscle cells, peripheral blood mononuclear cells, macrophages (including Kupffer cells), and HSCs are among the cells that express ALE receptors [95]. AGEs and ALEs are formed by Maillard reactions and share the same receptor, thereby eliciting similar responses. Activities associated with the ALE/AGE-RAGE pathway are reportedly increased in liver diseases [96][97][98][99][100]. Leung et al. demonstrated a higher deposition of CML in the liver of animals with NAFLD, induced by a diet deficient in methionine and choline, and an increase in the production of ROS by Kupffer cells dependent on the AGE-RAGE pathway [101]. Furthermore, a high intake of ALEs/AGEs has been shown to increase hepatic CML contents, thereby worsening liver damage and increasing levels of proinflammatory cytokines by activating HSCs in a RAGE pathway-dependent manner, which in turn leads to increased oxidative stress and fibrosis [102,103]. Increased oxidative stress and tissue inflammation caused by ALEs support the "multiple-hit hypothesis," suggesting that ALEs play a key role in the progression of simple steatosis to NASH and liver fibrosis. On the other hand, studies have shown that statins may act as negative regulators of RAGE and reduce ALEs [17], in addition to inducing an increase in sRAGE, a soluble form of RAGE that can act as an AGE/ALE scavenger [17]. Patients with hypercholesterolemia had lower levels of sRAGE than patients with normocholesterolemia, while plasma sRAGE levels in patients with statin-treated hypercholesterolemia were significantly higher than those in untreated individuals. In addition, sRAGE levels were inversely correlated with LDL levels (r = 0.36, p = 0.005) and total cholesterol levels (r = 0.326, p = 0.011) [104]. A study on type 2 diabetes demonstrated that atorvastatin reduced serum CML levels in hypercholesterolemic patients without any cardiovascular diseases. It was reported that blood glucose control levels were not altered by treatment with atorvastatin, and changes in CML were not correlated with those in lipid parameters [105], suggesting that statins may lower serum CML levels in a manner independent of its lowering of cholesterol.
Free radical nitric oxide (NO) plays an important role in the liver physiology and pathophysiology of NAFLD [106]. Many studies have discussed the vasoprotective role of NO generated by endothelial nitric oxide synthase (eNOS) in NAFLD; however, NO activity carries advantages as well as disadvantages. In the liver, small amounts of NO generated by eNOS are believed to promote liver homeostasis and protect against pathological conditions. By contrast, iNOS expression, which is absent in resting cells, is induced by immune stimuli and inflammatory cytokines during inflammation and plays a role in the development and maintenance of many liver diseases [106,107]. Moreover, proinflammatory cytokines, such as IL−1 and TNF-α, may induce iNOS expression in liver sinusoidal endothelial cells and hepatocytes [108][109][110]. NO is produced by activated HSCs as well, due to the action of iNOS [62,111,112]. Our findings confirmed that iNOS is overexpressed in the liver tissues of animals with NAFLD, together with an important increase in the NO metabolite, nitrite. Increased iNOS expression generates oxidative stress and leads to a proinflammatory condition that affects liver microcirculation [113,114]. SV treatment reduced iNOS expression and NO production. Statins decreased the activity of activating protein-1 (AP−1), a transcription factor that plays an important role in endothelial cell inflammatory responses and regulates genes responsible for cytokines, adhesion molecules, and iNOS production [115]. Statins negatively regulate the expression of iNOS in the endothelium and small kidney vessels of transgenic mice that express human renin and angiotensinogen [116], leading to increased eNOS activity and NO bioavailability, thereby preventing an increase in iNOS in rats after ischemia-reperfusion [117]. Furthermore, SV decreased iNOs and NO through decreased HSC activation [45].
Pearson's correlation analyses using NAFLD progression factors demonstrated that hepatic ALE levels were correlated with the modulation of key metabolic and microcirculatory parameters, including RAGE hepatic expression, liver basal microvascular blood flow, sinusoidal density, HSC activation, and leukocyte recruitment, which strengthens the hypothesis that advanced lipoxidation end products are strongly related to the pathophysiology of NAFLD and microcirculatory changes (Figure 6), in addition to being an important factor in the modulation of NAFLD complications. expressed in the liver tissues of animals with NAFLD, together with an important increase in the NO metabolite, nitrite. Increased iNOS expression generates oxidative stress and leads to a proinflammatory condition that affects liver microcirculation [113,114]. SV treatment reduced iNOS expression and NO production. Statins decreased the activity of activating protein-1 (AP−1), a transcription factor that plays an important role in endothelial cell inflammatory responses and regulates genes responsible for cytokines, adhesion molecules, and iNOS production [115]. Statins negatively regulate the expression of iNOS in the endothelium and small kidney vessels of transgenic mice that express human renin and angiotensinogen [116], leading to increased eNOS activity and NO bioavailability, thereby preventing an increase in iNOS in rats after ischemia-reperfusion [117]. Furthermore, SV decreased iNOs and NO through decreased HSC activation [45].
Pearson's correlation analyses using NAFLD progression factors demonstrated that hepatic ALE levels were correlated with the modulation of key metabolic and microcirculatory parameters, including RAGE hepatic expression, liver basal microvascular blood flow, sinusoidal density, HSC activation, and leukocyte recruitment, which strengthens the hypothesis that advanced lipoxidation end products are strongly related to the pathophysiology of NAFLD and microcirculatory changes (Figure 6), in addition to being an important factor in the modulation of NAFLD complications.

Conclusions
Our findings suggest that SV exerts pleiotropic effects, associated with negative modulation of lipid accumulation and peroxidation, ALE-RAGE pathway, oxidative stress, and iNOS expression, protects microcirculatory dysfunction associated with NAFLD, and recovers tissue perfusion, thereby contributing to the repositioning of statins for NAFLD treatment.